Our long-term goal is to identify genes that influence sensitivity to acute locomotor stimulation to ethanol, a putative mouse model of an endophenotype (behavioral stimulation) for alcoholism. The overall goal of this proposal is to determine if the m4 and/or m5 muscarinic acetylcholine receptor (mAChR) subtype genes have a role in acute locomotor stimulation to ethanol. Genes on mouse chromosome 2 (Chr 2) influence sensitivity to ethanol-induced stimulation;within the relevant region on Chr 2 reside the m4 and m5 mAChR subtype genes. The proposed studies are intended to elucidate if differences in m4 and/or m5 gene expression are associated with predisposition to ethanol-induced stimulation, and if they are directly involved in this behavior. The FAST and SLOW lines of mice were selectively bred for high and low sensitivity to the locomotor stimulant effects of ethanol, and are therefore a sensitive genetic animal model in which to study these differences. The specific aims of this proposal are to 1.) determine whether there are specific brain regions in which the m4 and/or m5 mAChR subtype genes are differentially expressed in FAST and SLOW mice, and 2.) determine whether selective gene silencing of the m4 and/or m5 mAChR subtype genes affects acute locomotor stimulation to ethanol. Gene expression will be assessed in lasercapture-dissected brain regions using qRT-PCR and Western blot analysis. Silencing of the m4 and m5 receptor subtype genes will be accomplished by microinjecting selective RNA interference (RNAi) transcripts into the nucleus accumbens (NAc) and ventral tegmental area (VTA), respectively. It is hypothesized that FAST mice will display decreased expression of the m4 gene in the NAc and enhanced expression of the m5 gene in the VTA as compared to SLOW mice. It is also hypothesized that microinjection of m4-selective RNAi into the NAc of SLOW mice will enhance the acute locomotor stimulant response to ethanol, while microinjection of m5-selective RNAi into the VTA of FAST mice will block ethanol-induced stimulation. It is important to understand the pharmacogenetic underpinnings of acute sensitivity to ethanol because it has some value for the prediction of future alcohol use/abuse. Findings from this proposal may inform future pharmacotherapies for development in the treatment of alcoholism.